Hoskins et al. Journal of Nanobiotechnology 2012, 10:15
http://www.jnanobiotechnology.com/content/10/1/15
RESEARCH
Open Access
The cytotoxicity of polycationic iron oxide
nanoparticles: Common endpoint assays and
alternative approaches for improved
understanding of cellular response mechanism
Clare Hoskins, Alfred Cuschieri and Lijun Wang*
Abstract
Background: Iron oxide magnetic nanoparticles (MNP’s) have an increasing number of biomedical applications. As
such in vitro characterisation is essential to ensure the bio-safety of these particles. Little is known on the cellular
interaction or effect on membrane integrity upon exposure to these MNPs. Here we synthesised Fe3O4 and surface
coated with poly(ethylenimine) (PEI) and poly(ethylene glycol) (PEG) to achieve particles of varying surface positive
charges and used them as model MNP’s to evaluate the relative utility and limitations of cellular assays commonly
applied for nanotoxicity assessment. An alternative approach, atomic force microscopy (AFM), was explored for the
analysis of membrane structure and cell morphology upon interacting with the MNPs. The particles were tested in
vitro on human SH-SY5Y, MCF-7 and U937 cell lines for reactive oxygen species (ROS) production and lipid
peroxidation (LPO), LDH leakage and their overall cytotoxic effect. These results were compared with AFM
topography imaging carried out on fixed cell lines.
Results: Successful particle synthesis and coating were characterised using FTIR, PCS, TEM and ICP. The particle size
from TEM was 30 nm (−16.9 mV) which increased to 40 nm (+55.6 mV) upon coating with PEI and subsequently
50 nm (+31.2 mV) with PEG coating. Both particles showed excellent stability not only at neutral pH but also in
acidic environment of pH 4.6 in the presence of sodium citrate. The higher surface charge MNP-PEI resulted in
increased cytotoxic effect and ROS production on all cell lines compared with the MNP-PEI-PEG. In general the
effect on the cell membrane integrity was observed only in SH-SY5Y and MCF-7 cells by MNP-PEI determined by
LDH leakage and LPO production. AFM topography images showed consistently that both the highly charged
MNP-PEI and the less charged MNP-PEI-PEG caused cell morphology changes possibly due to membrane
disruption and cytoskeleton remodelling.
Conclusions: Our findings indicate that common in vitro cell endpoint assays do not give detailed and complete
information on cellular state and it is essential to explore novel approaches and carry out more in-depth studies to
elucidate cellular response mechanism to magnetic nanoparticles.
Keywords: Magnetic nanoparticle, Cellular interaction, Cell membrane, Cytotoxicity, Cell viability assay, Atomic
force microscopy, Zeta potential
* Correspondence: [email protected]
Institute for Medical Science and Technology (IMSaT), Wilson House, 1
Wurzburg Loan, University of Dundee, Dundee DD2 1FD, UK
© 2012 Hoskins et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Hoskins et al. Journal of Nanobiotechnology 2012, 10:15
http://www.jnanobiotechnology.com/content/10/1/15
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Background
Recently magnetic nanoparticles have become the focus
of scientific interest due to their vast biomedical applications [1-3]. The solution instability and toxicity of iron
oxide nanoparticles have been extensively studied and
overcome by complete coating of the particles using
materials such as silica [4], polymers [5], inorganic
metals [6], bioactive molecules [7] etc. After coating the
MNP core the acute toxicity experienced is attributed to
the physicochemical properties of the particle surface
[8]. Such properties include hydrodynamic radius [9],
surface charge [10] and inherent toxicity of the coating
materials [11]. However, little is known of the mechanism of cellular interaction and the long term stability of
these particles in physiological conditions [12]. Cellular
fate is dependent on cellular responses to acute toxicity,
toxicity of degradation products and toxic effects due to
nanoparticulate systems [3]. As such as more applications for magnetic nanoparticles are realised priority
should be placed on the understanding of mechanisms
of nanoparticle-cell interaction and cellular response
that underline the toxicity of these particles.
It is presumed that after cellular uptake via endocytosis the clusters of iron oxide nanoparticles reside inside
lysosomes [13] followed by degradation into iron ions
via enzyme hydrolysis in the low pH environment [2]. It
has been reported that the reactive oxygen species
induced by transition metal particles can lead to lipid
peroxidation [14,15]. Lipid peroxidation results in the
disruption of the phospholipid bilayer membrane as a
result of intracellular stresses from hydrogen peroxide
production (Eqn. 1&2) [16,17]; this can also result in
cell mortality [18].
[O2 − + Fe3+ → O2 + Fe2+ ]
(1)
[2O2 − + 2H+ → O2 + H2 O2 ]
(2)
In 1987 Minotti and Aust investigated the requirement for iron (III) in the initiation of lipid peroxidation
[19]. Their findings suggested that lipid peroxidation
can only be initiated by the presence of both Fe2+ and
Fe3+ as alone neither Fe2+ nor Fe3+ could promote peroxidation of the lipid membrane [19]. This finding suggests that lipid peroxidation will occur in cells with
internalised Fe3O4 only if degradation of the particles
occurs.
The oxidation of Fe 2+ by H 2 O 2 believed to initiate
redox cycling promoting the free radical production via
the final step of the cycle, is known as Fenton’s reaction
(Eqn. 3) [2,19]. This free radical production causes intracellular stresses and can lead to cellular death [20,21].
Soenen et al. reported significantly increased free radical
production in C17.2 neural progenitor, PC12 rat
pheochromocytoma and human blood outgrowth cells
incubated with clinically available Endorem® and three
other iron oxide nanoparticles (Resovist®, magnetoliposomes and very small iron oxide nanoparticles) [11].
Although the stress levels were significantly greater than
control cells, Soenen et al. concluded that the contribution of nanoparticle-induced oxidative stress to the toxicity of these nanoparticles remained unclear as cells
possess inherent defence systems in order to deal with
varying oxidative stress levels [11].
[H2 O2 + Fe2+ → Fe3+ + OH− + OH∗ ]
(3)
In order to obtain a comprehensive safety profile of
iron oxide MNPs various studies should be carried out
which measure different aspects of the cellular response
[22]. Routine analysis for cytotoxicity of nanoparticles is
largely based on methods established for hazard characterisation of chemicals or cytotoxic drugs, using assays
such as the MTT (absorbance) or CellTiter Blue (fluorescence). As reported in several previous studies including our own [22-24], these commonly used endpoint
assays which measure cellular enzyme activity frequently
interact with nanoparticles and in our case, consistently
over-estimated cell viability when validated with traditional Trypan blue counting [24]. Commonly cytotoxicity data are used to evaluate the cellular fate after
exposure to magnetic nanoparticles; however these endpoint assays do not elucidate the cellular physiological
state. Cells impermeable to Trypan blue are assumed to
be viable and healthy; however, is this always the case
and to what extent do these in vitro studies reflect in
vivo conditions? Feridex is a dextran coated superparamagnetic iron oxide nanoparticle clinically administered
in MRI imaging of patients [25]. Although FDA
approved [25], Feridex still causes adverse reaction in
patients [26]. These reactions can lead to hypotension,
liver lesions, anaphylactic reaction which in severe cases
can be lethal [26,27]. The reasons for inter-patient sensitivities are not well understood. In order for future
novel metallic nanostructures to be safe for patient use
we believe understanding cellular state in response to
nanoparticle exposure is of utmost importance.
Atomic force microscopy (AFM) is an established
characterisation technique used for topographic imaging
especially in the physical sciences for materials such as
polymers [28], microchips [29] etc. With advances in
technology this powerful tool can now be applied to
biological samples [30]. The ability to obtain topography
images of cells allows for detailed cell morphology
visualisation which before was unobtainable [31]. Cell
membrane interaction with nanoparticles is a largely
unknown area. Studies have shown that upon cellular
incubation with nanoparticles nanosized pores develop
Hoskins et al. Journal of Nanobiotechnology 2012, 10:15
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in the cell membrane [32,33]. Vasir and colleagues
reported the use of AFM to image nanoscale holes in
the cell membrane after cellular exposure to copolymer
poly(D,L-lactide-co-gylcolide) coated iron oxide nanoparticles [33]. The noticed nanosized ‘pits’ appearing in
the cell membrane approximately 50 nm in depth and
170 nm in width at the surface. They postulated that
this could be due to the restructuring of the membrane
during the initial phase of endocytosis [33].
Here we will synthesise magnetic Fe3O4 nanoparticles
and coat with poly(ethylenimine) (PEI) and subsequently
poly(ethylene glycol) (PEG) giving rise to differing surface positive charges. PEI is a common polycation used
frequently for coating magnetic nanoparticles, drug
delivery and as a transfection agent [34]. PEG is a
hydrophilic polymer used frequently in the coating of
magnetic nanoparticles, it possess’ many desirable qualities such as increased biocompatibility and ‘stealth’
properties leading to increased circulation times [34].
After surface coating we will carry out in vitro biocompatibility studies and explore the potential of AFM in
elucidating nanoparticles-cell membrane interactions
using three human cell lines including neuroblastoma
(SH-SY5Y), breast cancer (MCF-7) and macrophage-like
(differentiated U937) cells.
Page 3 of 11
polymer even after 8 h freeze drying. The distinction
between MNP-PEI and its pegylated counterpart was
made via the absence of the primary amine peaks at
3300, 1700 &1600 cm -1 which were observed in the
MNP-PEI sample. The alkyl peak at 2800 cm-1 appeared
more pronounced in the presence of the PEG moiety
due to the nature of the polymer backbone. Additionally
a small peak was observed at 3400 cm-1 which was due
to the C = O stretch of the bonds in the PEG moiety
(Figure 1). Inductively coupled plasma (ICP) spectroscopy was used to deduce the concentration based on
the total iron content of the MNPs. The ‘naked’ MNPs
had a hydrodynamic radius of 1112 nm determined by
photon correlation spectroscopy (Figure 2A). This large
value indicated that aggregation of the individual particles had occurred due to their inherent magnetic properties, hence a large polydispersity index was observed
(0.763) (Figure 2B). The TEM micrograph gave a more
realistic representation of the MNP size which was
approximately 30 nm (Figure 2C1). After PEI coating
the concentration was 13.5 mgmL-1 (93% yield) and 11.1
mgmL -1 (82% yield) after subsequent pegylation. The
polymer coated nanoparticles appeared more stable in
solution and aggregation was reduced with the hydrodynamic radius for both MNP-PEI and MNP-PEI-PEG
Results
Synthesis and characterisation of MNPS
The particles were synthesized and coated with PEI and
PEG. Fourier transform infrared spectroscopy (FTIR)
analysis of the freeze dried particles confirmed the
attachment of polymer backbone to the ‘naked’ particles
with the presence of -NH peaks at 3300, 1700 & 1600
cm-1 and a distinct C-N peak at 1000 cm-1 (Figure 1).
The broad peak observed at 3100 cm-1 was due to free
water which was still present in this hygroscopic
Figure 1 FTIR spectra of freeze dried MNP (red), MNP-PEI
(blue) and MNP-PEI-PEG (purple) carried out on a Nicolet IS5
with and ID5 diamond tip ATR attachment. 64 scans were
carried out for each sample.
Figure 2 Size estimations of MNPs analysed by A) Photon
correlation spectroscopy showing the surface charge and B)
Polydispersity index of particles measured at 1 mgmL-1 in
deionised water (n = 9 ± SD) and C) TEM images of 1) naked
MNP, 2) MNP-PEI and 3) MNP-PEI-PEG.
Hoskins et al. Journal of Nanobiotechnology 2012, 10:15
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significantly reducing to 237 nm and 262 nm respectively (p < 0.005) (Figure 2A). The surface charge of the
MNPs was determined by zeta potential measurement.
The ‘naked’ MNP’s possessed a negative surface charge
(−16.9 mV) due to sulphate associations previously
reported to result from the synthetic route [35] (Figure
2A). Addition of PEI increased the overall zeta potential
measurement to +55.6 mV due to the positive charge
on the amine groups of the polymer backbone; this
measurement gives good indication that coating was
successful. The zeta potential measurement for the
pegylated particle was +31.2 mV. The decrease observed
is attributed to the presence of -OH groups on the PEG
coating as previously reported [24].
Stability of coated MNPs
The stability of MNPs against degradation at pH’s
mimicking physiological (7.2) and intracellular (4.6 and
4.6 with 20 mM citrate ions) environments were determined over a 2 week period (Figure 3). The results were
expressed as a percentage weight of the initial starting
MNP iron weight (200 µg). At pH 7.2 a maximum of
0.04% of the initial iron concentration was observed in
the media for the MNP-PEI and its pegylated counterpart. The release rate for both MNPs appeared to be
consistent over the time period. At pH 4.6 particle
degradation appeared to be greater. An initial burst
release of iron (0.027%) was observed in the first 24 h
followed by a slow incline over the duration to a maximum of 0.055% and 0.049% for MNP-PEI and MNPPEI-PEG respectively. The increased degradation compared with at higher pH can be attributed to the iron
core being slowly dissolved in acidic environments.
Upon addition of citrate ions into pH 4.6 media the
iron degradation increased significantly after 1 week (p
< 0.005), and up to a maximum of 0.149% and 0.145%
Page 4 of 11
for MNP-PEI and MNP-PEI-PEG respectively, in two
week time. Citrate ions possess the ability to chelate
with the iron molecules leading to hydrolysis and resulting in particle degradation [36]. In general, the MNPPEI appeared to degrade slightly more when compared
to the MNP-PEI-PEG; however this was not significant
(p > 0.005). Based on the amount of released iron ion,
the stability of our synthesized MNP’s is comparable
with that of clinically approved iron oxide nanoparticles
ferumoxides [25] as shown by a previous study [37].
Cellular uptake of nanoparticles
The intracellular content of MNPs in SH-SY5Y, MCF-7
and U937 cells was determined by ICP after incubation
for 1, 4, 24 and 72 h with 25 µgmL-1 MNPs (Table 1).
The MNP cellular uptake appeared to be time dependant
up to 24 h for both the MNP-PEI and MNP-PEI-PEG for
in all cell lines. After 24 h the cellular uptake appeared to
plateau indicating that the rate of cellular uptake was
greatest within this time. The U937 cells resulted in
lower intracellular iron levels after 72 h compared with
the SH-SY5Y and MCF-7 cells, this finding is interesting
when considering the phagocytotic nature of these cells.
However, the relatively smaller cell volume of the U937
cells may result in a lower maximum uptake compared
with the larger SH-SY5Y and MCF-7 cells.
Cell viability by nanoparticle exposure
Viability in response to nanoparticles was evaluated by
Trypan blue exclusion for cells exposed to nanoparticles
over 7 days (Figure 4). After 24 h incubation with
MNP-PEI up to 30%, 50% and 20% reduction in viability
was observed in SH-SY5Y, MCF-7 and U937 cell lines
respectively at 100 µgmL-1. However, the MNP-PEI-PEG
experienced only a maximum of 5% viability reduction
across the three cell lines. After 168 h the viability
dropped to 10%, 20% and 30% for MNP-PEI incubated
cells compared with around 90% for the MNP-PEIPEG’s in all cells.
Cell membrane integrity after incubated with
nanoparticles
Figure 3 Stability of 1)MNP-PEI (open marker) and 2) MNP-PEIPEG (filled marker) in RPMI-1640 media of pH 7.2 (◇), pH 4.6
(○) and pH 4.6 containing sodium citrate (20 mM) (□). Study
carried out using 2 mL, 100 μgmL-1 MNPs under sink
conditions with stirring over 2wks (n = 3 ± SD).
The cell membrane integrity was determined via quantification of the LDH leakage from cells incubated with
nanoparticles compared to control cells. In SH-SY5Y
cells (Table 2) there was no increase from the basal
values (10%) at all MNP-PEI concentrations during
short incubation periods (1 h to 24 h) except at the
highest concentration (100 µgmL-1) at 24 h. After 72 h
incubation a significant increase (30%, p < 0.005) in
LDH leakage was observed across the whole concentration range. The MCF-7 cells experienced an increase in
LDH leakage after 24 and 72 h incubation with MNPPEI (p < 0.005) (Additional file 1: Table S1). Similar to
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Table 1 Cellular uptake of polymer coated MNP in SH-SY5Y, MCF-7 and U973 cells at 25 µgmL-1 over 72 h (n = 3 ±
SD).
Concentration of Fe3+ uptake per cell, pg (± SE)
Particle
Incubation time h
SH-SY5Y
MCF-7
U937
MNP-PEI
0
0.560 (0.017)
0.261 (0.004)
0.121 (0.087)
1
16.205(0.867)
7.990 (3.581)
7.017 (0.377)
4
14.426 (0.577)
14.257 (0.415)
11.640 (0.405)
24
23.277 (0.506)
16.167 (1.258)
12.057 (1.315)
72
25.580 (0.353)
19.847 (1.305)
13.803 (1.842)
MNP-PEI-PEG
0
0.560 (0.017)
0.261 (0.004)
0.121 (0.087)
1
8.227 (0.523)
7.027 (0.424)
5.163 (0.484)
4
9.997 (0.451)
9.963 (0.791)
7.353 (0.380)
24
17.770 (1.462)
14.257 (0.297)
9.740 (0.986)
72
18.700 (0.360)
16.593 (0.756)
9.627 (0.997)
the SH-SY5Y cells a time dependant trend was observed
irrespective of concentration. No increase from basal
levels (6-7%) of LDH leakage was observed in the U937
macrophage-like cells. (Additional file 1: Table 2). In
general the MNP-PEI-PEG did not cause LDH leakage
in all three cell lines.
Cellular oxidative stress measured by reactive oxygen
species (ROS) production and lipid peroxidation
The ROS assay determines the intracellular stress levels
due to the production of free radical oxygen species. For
all three cell types tested the MNP-PEI caused significant increase in free radical production. In SH-SY5Y
cells (Table 3) the PEI coated particles resulted in up to
20% increased stress levels compared with the control
cells. The MNP-PEI-PEG did not appear to increase the
oxidative stress levels in line with our previous findings
[24]. The MCF-7 cells (Additional file 1: Table S3)
appeared to experience much larger stress levels with up
to 54% increase compared to the control cells when
incubated with MNP-PEI. Again the pegylated particle
did not appear to cause any stress due to free radical
production. Similar to the SH-SY5Y cells, the U937 cells
(Additional file 1: Table S4) experienced up to 10% elevated stress levels due to the MNP-PEI with no significant increase upon incubation with MNP-PEI-PEG.
Hydrogen peroxide generation can occur inside cells as
a secondary product from reactive oxygen species. The
presence of these species can initiate stress to the cell
membrane in the form of lipid peroxidation [19]. The
results from the TBARS assay in SH-SY5Y cells can be
seen in Table 3. These indicated that the MNP-PEI and
MNP-PEI-PEG did not appear to result in any significant induction of lipid peroxidation in all three cell lines
(MCF-7 Additional file 1: Table S3 and U937 Additional
file 1: Table S4) tested when compared to control cells.
AFM topography imaging of MNP - cellular interactions
and cell membrane roughness analysis
Figure 4 Trypan blue exclusion assay using A) Fe3O4-PEI and
B) Fe3o4-PEI-PEG on 1) SH-SY5Y, 2) MCF-7 and 3) U937 cells
over □ 24, ■ 72, ■120 and ■168 h (n = 3 ± SD).
In order to determine whether the cellular assays provided a good indication of the overall physiological state
of the cell AFM topography images were obtained. Figure 5 shows the topography images for fixed SH-SY5Y
cells, only one cell is shown for each time point (1, 4,
24, 72 h); however in practice extensive cellular images
were obtained. These cells are shown as a representative
of the overall cell state. The control cells (Figure 1, 2, 3,
4) appeared smooth and well formed with definite cell
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Page 6 of 11
Table 2 Percentage cytotoxicity on cell membrane measured via LDH leakage using MNP-PEI and MNP-PEI-PEG on SHSY5Y cells over 1, 4, 24 and 72 h (n = 3 ± SD)
MNP concentration µgmL-1
Particle
Incubation time h
0
6.25
12.5
25
50
100
MNP-PEI
1
10.29 (0.607)
12.89 (0.282)
12.15 (0.152)
12.16 (0.357)
12.66 (0.952)
14.42 (0.997)
4
9.73 (0.014)
13.30 (1.052)
13.21 (1.192)
12.38(0.183)
12.16 (0.339)
12.35 (0.428)
24
10.26 (0.014)
10.75 (0.080)
11.07 (0.344)
11.38 (0.021)
13.90 (0.132)
24.94 (0.789)*
72
11.86 (0.288)
32.43 (2.366)*
35.51 (1.897)*
33.03 (2.103)*
30.86 (1.464)*
31.37 (0.788)*
1
10.44 (0.007)
10.43 (0.526)
9.95 (0.120)
9.85 (0.010)
9.80 (0.288)
9.93 (0.087)
4
10.52 (1.539)
11.61 (0.616)
11.66 (0.158)
11.62 (0.350)
11.71 (0.102)
11.63 (0.484)
24
10.35 (1.531)
9.02 (0.027)
8.67 (0.153)
8.84 (0.072)
9.00 (0.024)
9.50 (0.295)
72
11.13 (0.242)
11.29 (0.591)
11.17 (0.443)
12.11 (2.964)
10.69 (0.254)
12.98 (0.000)
MNP-PEI-PEG
* Denotes a significant increase from basal levels (p < 0.05).
boundaries. This appearance was consistent throughout
all the incubation times (1-72 h). After addition of the
positively charged PEI-coated particles the cells
appeared increasingly flattened and less structured.
After 24 h incubation with MNP-PEI the cell morphology appeared to have completely changed. Interestingly,
upon incubation with the MNP-PEI-PEG a similar trend
was observed. Cell flattening had begun after only 4 h
incubation with the particles andafter 72 h more dramatic change in cell surface topography was observed. A
similar phenomenon was observed when both MNP-PEI
and MNP-PEI-PEG were incubated with MCF-7 cells
(Additional file 1: S1, Figure 1). With U937 the cell
deformation appeared to be much smaller than that of
SH-SY5Y and MCF cells (Additional file 1: S1, Figure
2). In all cells the MNP exposure appeared to cause
small pits in the cell membrane resulting in a rougher
cell surface. These results of morphological alteration
represent a different pattern of cellular responses to
iron oxide nanoparticles. These findings were in
Table 3 ROS (% of control cell) and LPO induction by
MNPs in SH-SY5Y cells incubated with 25 µgmL-1 for 1, 4,
24 and 72 h (n = 3 ± SD).
Particle
Incubation
time h
ROS Assay
LPO Assay
% DCF
MDA nM/mg protein
fluorescence (Control cells: 2.620 ±
0.225)
MNP-PEI
MNP-PEIPEG
1
99.00 (5.568)
2.702 (0.015)*
4
115.67
(5.033)*
2.567 (0.188)
24
121.67
(7.371)*
2.667 (0.321)
72
113.67
(4.509)*
2.638 (0.157)
1
97.33 (6.658)
2.282 (0.341)
4
101.00
(5.292)
2.383 (0.018)
24
103.33
(3.512)
2.383 (0.299)
72
102.00
(2.000)
2.651 (0.107)
* Denotes a significant increase from basal levels (p < 0.05).
Figure 5 AFM topography images of SH-SY5Y cells. A) control
cells without MNPs, B) cells incubated with 25 µgmL-1 MNP-PEI
and C) MNP-PEI-PEG over 1) 1 h, 2) 4 h, 3) 24 h and 4) 72 h.
Cells were fixed after incubation and AFM imaging was performed
in air using a RTESPA tip of spring constant 40 N/m, carrying out
896 scans/line at a scan rate of 0.32 Hz and 1.102 V amplitude.
Hoskins et al. Journal of Nanobiotechnology 2012, 10:15
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agreement in part with the cell membrane integrity
measured via LDH leakage where a time dependant
membrane disruption by MNP-PEI was observed and
that MNP-PEI did not caused significant cell membrane
damage in U973 cells (Table 2, Additional file 1: Table
S1 and S2). These data also partially matched the level
of the cellular oxidative stress in terms of ROS production by MNP-PEI (Table 3, Additional file 1: Table S3
and S4) where the increase in ROS reached its peak
after 24 h incubation of MNP-PEI. As MNP-PEI-PEG
were shown to induce neither cell membrane disruption
nor cellular oxidative stress in all cells tested (Table 2,
3, Additional file 1: Tables S1, S2, S3, S4), the relationship between cell morphological change due to the
interaction between MNPs and cells and measured cell
parameters such as membrane integrity and oxidative
stress are yet to be established. It should be noted that
these traditional assays are endpoint measurements (and
Trypan blue) and do not give an indication on the cellular dynamic physiology state. Perhaps AFM can be used
as an important tool for assessing the biological effect
these metallic nanostructures have in vitro which cannot
be elicited by standard cell biological techniques.
The cell surface roughness analysis data on SH-SY5Y
cells (Figure 6) further indicated that the cell membrane
was affected by nanoparticle incubation [33]. These data
are relative to control cells and serve only as a guide to
describe the whole cellular state. In SH-SY5Y cells incubated with MNP-PEI the membrane roughness had virtually doubled after only 1 h. After 24 h incubation a
2.2-fold increase had occurred, however, after 72 h the
roughness appeared to decrease slightly. These observations correlate with the level of MNP-PEI-induced ROS
in which the largest increase was observed at 24 h incubation (Table 3, Additional file 1: Table S3, S4). This
could also be a result of flattening of cells or adaptation
of the cellular cytoskeleton in response to nanoparticle
Figure 6 Roughness analysis carried out on fixed SH-SY5Y cells
of AFM topography images and analysed using NanoScope
Analysis software (n = 3 ± SE). * Denotes a significant increase
compared to control cells (p < 0.05).
Page 7 of 11
encounter. The decreased cell roughness with time was
however not significant (p > 0.05). Similar to the MNPPEI the cells that were incubated with MNP-PEI-PEG
experienced an increase in membrane roughness over
the duration of incubation. A decrease in membrane
roughness compared to the 24 h values (p > 0.05)
occurred after 72 h consistent with the MNP-PEI.
Discussion
In this study we successfully synthesised magnetic iron
oxide nanoparticles. The nanoparticles appeared to be
mono-dispersed and around 30 nm in size (Figure 2C1).
Polymer coating was achieved with both PEI and PEG
which was confirmed with FTIR and zeta potential measurement. The MNPs appeared to be stable in conditions mimicking cellular pH up to 2 weeks with less
than 0.15% of iron being released (Figure 3).
Increased cellular uptake was observed in the MNPPEI compared with MNP-PEI-PEG which was attributed
to the higher positive surface charge (+55.6 mV) attracting to the negative cell membrane and enhancing endocytosis [35]. The cell viability data (Figure 4) showed
clearly that after pegylation of the MNP-PEI the cytotoxicity was significantly reduced (p < 0.005) in line
with our previous findings [24]. The primary amines on
the surface of the MNP-PEI give rise to the large positive surface charge (55.6 mV) and have previously been
reported to cause a cytotoxic effect [38]. The reduction
in cytotoxicity observed in the pegylated particles arises
due to the decreased surface charge [38] and ‘stealth’
properties [34] on the particle surface. The highly
charged MNP-PEI showed a concentration independent
and time dependent effect on the LDH leakage from the
SH-SY5Y and MCF-7 cells (Table 2 and Additional file
1: Table S1 respectively); this trend was not observed
with the MNP-PEI-PEG with reduced surface charge
where no deviation from the basal level was evident.
The concept of Trypan blue exclusion and LDH leakage
is similar however, the exact mechanism and molecular
cut-off points for each molecule to pass the cell membrane is unknown. Perhaps this can explain the contrasting LDH leakage results where the cytotoxic effect
is concentration independent compared with the Trypan
blue exclusion.
In line with our previous study the MNP-PEI significantly (p < 0.005) increased ROS production resulting in
cellular stress. After pegylation the stealth quality of the
MNPs resulted in free radical production consistent
with the control cells (Table 3) [24]. This result coupled
with the stability data indicated that the free radical
increase with MNP-PEI was likely to be caused by the
increased positive charge on the polymer backbone and
hence possible disruption of endosomal organelles [39]
and not from the release of iron in the cytoplasm. Both
Hoskins et al. Journal of Nanobiotechnology 2012, 10:15
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MNPs showed no significant increase in lipid peroxidation (Table 3) suggesting that lipid peroxidation is not
the major cause of cytotoxicity or other aspect of lipid
oxidative stress not measured in this study was involved.
AFM topography imaging on fixed cells consistently
showed that cellular morphology was dramatically
altered after incubation with the MNPs (Figure 5 and
Additional file 1: Figure 1). The greater cell topographical change in the SH-SY5Y cells perhaps could be
attributed to the greater concentration of intracellular
nanoparticles (Table 1). The U937 cells showed the
smallest membrane structure change possibly due to
their specialised functionality as phagocytic and scavenger cells and thus having a stronger cell defence capacity, as evidenced also by virtually no LDH leakage and
very small increase in ROS production by MNP-PEI
(Additional file 1: Table S2 and S4). The cell morphological observation by AFM in the SH-SY5Y and MCF7
cells was also in partial agreement with the level of the
cellular oxidative stress (Table 3, Additional file 1: Table
S3) where the increase in ROS reached its peak after 24
h incubation of MNP-PEI. Therefore, the link between
cellular oxidative stress and cell morphological or other
physical properties which could aid in improving our
understanding of MNP toxicity and establishing more
reliable methodology in toxicity evaluation, merits
further investigation. Surprisingly, cells incubated with
the lower charged MNP-PEI-PEG responded to nanoparticle exposure in a similar manner to the MNP-PEI,
although no corresponding increase in LDH leakage and
oxidative stress was found. The real nature of cell membrane morphology change during contact with nanostructures may be accredited to endocytosis [33] as well
as possible membrane disruption by potential quantum
mechanical effect and other nano-activities. Dissection
of endocytosis-specific and “nano"-specific mechanisms
underlying the change in cell membrane topography is
currently undergoing in our lab.
As different aspects of MNP toxicity could contribute
to their overall biological effect [22], the endpoint cytotoxicity, as judged in this study by Trypan blue exclusion, could be attributed to a complex combination of
various factors, oxidative stress and cell membrane disruption being one of them. This is particularly important in the consideration of cell type-dependent
responses, such as epithelial versus phagocytic immune
cells, as immune cells (human macrophage-like U973
cells), could produce significant amount of cytokines in
response to nanoparticles which in turn would greatly
enhance the toxicity of nanoparticles under static cell
culture conditions [40]. This may partially explain the
negative membrane disruption and very small oxidative
stress response and yet comparable (to the SH-SY5Y
and MCF7 cell) overall cytotoxicity by MNP-PEI in
Page 8 of 11
U937 cells (Additional file 1: Table S2 and S4, and Figure 4).
Conclusion
Our data indicates that the kinetics in cell morphology
change resulting from iron oxide nanoparticle exposure
may reflect a different aspect of cellular stress compared
to those measured by conventional endpoint cell toxicity
assays. As such we propose that these commonly used
endpoint assays should not be used solely in determination of the safety profile of novel nanomaterials. In
order to fully understand these observations more work
needs to be carried out with regard to the cell membrane property and reorganisation of cytoskeletal system
and alteration of other cellular properties in response to
nanoparticles.
Methods
All chemicals were purchased from Sigma-Aldrich
unless otherwise stated.
Synthesis of Fe304 nanoparticles
The synthesis was based on the established protocol of
oxidative hydrolysis, i.e., the precipitation of an iron salt
(FeSO 4 ) in basic media (NaOH) with a mild oxidant
[41]. In brief, nitrogen was bubbled through a solution
of sodium hydroxide (0.1 M) and potassium nitrate (0.1
M) dissolved in deionised water at 90°C for 1 h. Iron
sulphate (0.03 M) dissolved in sulphuric acid (0.01 M)
was added to the reaction and the mixture was stirred
for 24 h at 90°C under nitrogen. After this time the
reaction was rapidly cooled on ice and the particles
were washed X6 deionised water and magnetically separated from solution. The resultant particles were re-suspended in water and stored at 4°C.
Coating and characterisation of MNPs
Iron oxide MNPS (2 mL) were sonicated in poly(ethylenimine) solution (5 mgmL-1) for 2 h. The particles were
then washed X6 with deionised water and magnetically
separated from solution. The MNP-PEI’s were resuspended in 10 mL deionised water and stored at 4°C.
MNP-PEI were added to 0.08 M sodium tetraborate followed by addition of methoxypolyethylene glycol pnitrophenyl carbonate (mPEG, MW 5000) (20 mg) with
stirring for 3 h at 22°C in the absence of light. The
resultant solution was washed with deionised water and
the MNP-PEI-PEG’s eluted from solution using a high
powered magnet. The MNP-PEI-PEG’s were resuspended in deionised water and stored at 4°C. Freeze
dried particles were run on the FTIR (Nicolet IS5 & ID5
ATR attachment, Thermo Scientific, UK) to determine
whether polymer coating was successful. Nanoparticle
concentration was determined using ICP analysis
Hoskins et al. Journal of Nanobiotechnology 2012, 10:15
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Page 9 of 11
(Optima 7000 V DV, Perkin Elmer, UK). The particles
were dispersed in deionised water and sonicated for 10
min before all measurements. Hydrodynamic diameters,
polydispersity index and zeta potential measurements
were carried out using a photon correlation spectrometer (Zetasizer Nano-ZS, Malvern Instruments, UK).
All measurements were conducted in triplicate at 25°C
and an average value was determined. Prior to zeta
potential analysis standard control samples were run on
the instrument.
water and run on the ICP (Optima 7000 V DV, Perkin
Elmer, UK). A calibration was carried out using iron
standard solutions 0.05 - 10 ugmL -1 (R = 0.9999). A
control sample of deionised water was also run.
*Differentiated U937 cells were used for all subsequent
experiments to represent human macrophage-like cell
conditions. Cells were differentiated by incubating cells
with 10 nM Phorbol 12-myristate 13-acetate (TPA) for
3 days, followed by 1 day with fresh media prior to all
experiments.
Degradation stability of coated MNPs
Cell viability assay
The stability of MNPs was evaluated based on established method [37] in RPMI-1640 media (Invitrogen,
UK) with pH’s representative of physiological (7.2) and
intracellular (4.6) environments. Sodium citrate (20
mM) was added in pH 4.6 media to further mimic
endosomal conditions. MNP solutions (2 mL, 100
µgmL -1 ) were placed inside dialysis membrane with
molecular cut-off 12-14 KDa. The dialysis tubes were
placed inside large conical flasks and stirred in 200 mL
of appropriate media under ‘sink’ conditions. At
1,4,72,168 and 336 h a sample of media was removed
(500 µL) and replaced with equal volume fresh media of
similar pH. Sample media (100 µL) was added to 900 µL
deionised water in an eppendorf tube. To each sample
(1 mL), 4.95 mM bathophenanthroline disulfonic acid
(40 µL) was added. The absorbance was measured after
90 s incubation at 535 nm (Techan M200 microplate
reader). The samples were finally incubated with 100
mM ascorbate solution for 8 min before a final absorbance measurement was conducted at 535 nm. The final
absorbance value was calculated as the positive difference between the initial reading subtracted from the
final reading. The concentration of free Fe3+ was calculated with respect to a standard curve (R 2 = 0.9943).
The total free Fe3+ was calculated as a percentage (w/w)
in respect to the starting amount.
Cell viability was determined using Trypan blue exclusion (Invitrogen, UK). Briefly SH-SY5Y, MCF-7 and
U937 cells were seeded in a 12 well plate and incubated
for 24 h at 37°C with 5% CO2 . The cells were treated
with increasing concentrations of MNP solutions (6.25 100 µgmL-1) and incubated for 24, 72, 120 and 168 h.
The cells were washed with PBS x3 and trypsinised.
Trypan blue was added to 100 µL cell suspension in
equal volume and incubated for 5 min at room temperature. The viable cells were counted. Values of viability of
treated cells were expressed as percentage of that from
corresponding control cells. All experiments were
repeated at least three times.
Cellular uptake of nanoparticles measured by inductively
coupled plasma (ICP)
SH-SY5Y, MCF-7 and U937* cells (ATCC, USA) seeded
in 6-well plates and incubated with MNPs (25 µgmL-1)
for 1,4, 24 and 72 h. The cells were washed X3 with
PBS, trypsinised (Invitrogen, UK) and re-suspended in
medium (Invitrogen, UK). The cell number was counted
using a Countess™ Automated Cell Counter (Invitrogen, UK) and cells were placed in eppendorf tubes (1 ×
106 cells/tube). The cell suspensions were centrifuged at
800 rpm for 5 min and the supernatant discarded. Concentrated hydrochloric acid (100 µL) was added to the
cells and the tubes were incubated at 90°C for 0.5 h.
The samples were cooled and centrifuged at 1500 rpm
for 10 min. The supernatant was diluted with deionised
Cell membrane integrity assay
Membrane integrity was measured via measurement of
lactate dehydrogenase (LDH) leakage (Promega, UK)
from SH-SY5Y, MCF-7 and U937 cells. Cells were
seeded into a 96-well plate (10,000/well) and incubated
for 24 h. The medium was replaced with increasing
magnetic nanoparticles concentrations (6.25 - 100
µgmL-1). The plates where incubated for 1, 4, 24 and 72
h. Lysis buffer (2 µL) was added to positive control
wells and the plate was centrifuged at 1500 rpm for 10
min at 37°C. The supernatant (50 µL) was then placed
into a new plate and equal volume of membrane integrity assay reagent was added. The plates were incubated
for 10 min at 37°C protected from light. 25 µL stop
reagent was then added to the wells and the fluorescence of the samples was measured at 560 nm (excitation) and 590 nm (emission) on a Techan M200
microplate reader. The percentage of cytotoxicity in
respect to the positive control wells was calculated
whereby the lysed cells were assumed to have 100%
LDH release.
Reactive oxygen species (ROS) assay
SH-SY5Y, MCF-7 and U937 cells were seeded into a 96well plate (10, 000/well) and incubated for 24 h. Cells
were incubated with increasing MNP concentrations
(6.25 - 100 µgmL-1) for 1, 4, 24 and 72 hrs. The cells
were washed 3X with PBS and incubated for 1 h with
Hoskins et al. Journal of Nanobiotechnology 2012, 10:15
http://www.jnanobiotechnology.com/content/10/1/15
100 µM carboxy-H2DCFDA (Invitrogen, UK) in PBS at
37°C protected from light. The cells were washed 3X
with PBS and incubated with serum free medium (100
µL) for 0.5 h. The medium was removed and replaced
with PBS. The fluorescence intensity of the samples was
measured at 560 nm (excitation) and 590 nm (emission)
on a Techan M200 microplate reader. The percentage
of DCF fluorescence was calculated in respect to control
cells assumed to be 100%.
Lipid peroxidation measurement by thiobarbituric acid
reactive substance (TBARS) assay
SH-SY5Y, MCF-7 and U937 cells were seeded into a 6well plate and incubated for 24 h. The medium was
replaced with increasing MNP concentrations (6.25 100 µgmL-1) and cells were incubated for 1, 4, 24 and
72 h. The cells were washed 3X with PBS and trypsinised. The cells were resuspended in 0.5 mL PBS containing 0.05% butylated hydroxytoluene. The cell
suspensions were sonicated for 5 s 3X at 40 V and kept
on ice. Malondialdehyde bis(dimethyl acetal) (MDA)
standard solutions (0-5 µM) were prepared and 100 µL
of samples or standards were added to Eppendorf tubes.
Sodium dodecyl sulphate (SDS) (100 µL, 2%) was added
and the tubes were incubated for 5 min at room temperature. Thiobarbituric acid (250 µL) was added to the
eppendorf tubes before incubation at 95°C for 1 h. The
samples were cooled on ice and centrifuged at 3000
rpm for 15 min at 4°C. The supernatant was pipetted
into the wells of a 96 well plate and fluorescent measurements were taken at 530 nm (excitation) and 550
nm (emission). The results were calculated as nmol of
MDA/mg of cellular protein.
Protein content was determined by addition of 100 µL
sample to 3 mL bradford reagent. The samples were
mixed well at room temperature for 5 min and absorbance was measured at 595 nm. The absorbance values
were compared to a calibration curve carried out using
bovine serum albumin and the protein concentration
was determined.
AFM topography imaging of MNP - cellular interactions
SH-SY5Y, MCF-7 and U937 cells were seeded in 6-well
plates containing collagen coated glass coverslips (SHSY5Y cells used non-coated coverslips). Cells were incubated for 24 h at 37°C and 5% CO2. MNPs (25 µgmL-1)
were added to the cells and further incubated for 1,4,24
and 72 h. Cells were washed X3 with PBS and fixed
with 2.5% gluteraldehyde in PBS for 10 min. Fixed cells
were washed X3 with deionised water and mounted on
glass slides. Cell topography imaging was carried out
using BioScope Catalyst AFM (Bruker, Germany) with
ScanAsyst Adaptive Mode. Cell membrane topography
was imaged using an RTESPA tip of spring constant 40
Page 10 of 11
N/m, carrying out 896 scans/line at a scan rate of 0.32
Hz and 1.102 V amplitude. At least three cells were
imaged to give a fair representation of each sample
condition.
Cell membrane roughness analysis
Cell membrane roughness was measured on the topography images using Nanoscope Analysis software (Bruker, Germany). Small areas (870 × 870 nm) were chosen
at random on ten areas of each cell and their membrane
roughness determined. An average was calculated from
a total of thirty areas from three cells.
Additional material
Additional file 1: Supplementary data. Figures 1 and 2 and Tables S1S4.
Acknowledgements
This work was financially supported by the MARVENE (ERA-NET NanoSci-E +
NAN092) EU project and the Engineering and Physical Sciences Research
Council (EPSRC) UK (EP/H007040/1 to LW and EP/H010033/1 to AC). We
wish to thank Dr Zhigang Wang and Mr Dun Liu for their valuable support
in carrying out the AFM study. All TEM images were carried out by Mr John
James in College of Life Sciences, University of Dundee. ICP studies were
carried out in School of Pharmacy and Life Sciences, Robert Gordon
University, Aberdeen. Photon Correlation spectroscopy and zeta potential
measurements were carried out in Dr Pascal Andre’s lab in School of Physics
and Astronomy, St. Andrews University.
Authors’ contributions
CH carried out coating, characterisation, cell and AFM experiments, and
wrote the paper. LY supervised the work and corrected the manuscript. AC
was a scientific advisor and edited the manuscript. All authors read and
approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 6 October 2011 Accepted: 17 April 2012
Published: 17 April 2012
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Cite this article as: Hoskins et al.: The cytotoxicity of polycationic iron
oxide nanoparticles: Common endpoint assays and alternative
approaches for improved understanding of cellular response
mechanism. Journal of Nanobiotechnology 2012 10:15.
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